1. Field of the Invention
This invention relates to nucleic acid probes useful for differentiating two closely related species of Campylobacter, C. Fetus, and C. hyointestinalis.
The genus Campylobacter is composed of spirally curved gram negative pathogens with characteristic darting motility but few metabolic characteristics useful in species differentiation. The genus encompasses pathogens of human and veterinary importance, including Campylobacter fetus and Campylobacter hyointestinalis. A vibroid bacterium, Vibrio fetus, has been recognized as the etiological agent of bovine and ovine infertility and abortion. This pathogen has been isolated from the placenta of aborting sheep, stomach contents of aborted fetuses and from blood, and also intestinal contents of infected ewes and cattle. The abortifacient species C. fetus was subsequently subdivided into two subspecies which are differentiated according to their tolerance to 5% glycine. C. fetus subsp fetus is transmitted orally, induces abortion in sheep, and rarely produces septicemia in humans; whereas C. fetus subsp venerealis is exclusively a venereal pathogen of animals. C. fetus subsp fetus replicates at 42.degree. C. and grows in the presence of glycine, whereas the subspecies venerealis replicates at 37.degree. C. and is intolerant of glycine [Smibert, "Genus Campylobacter Seabld and Veron 1963, 907.sup.AL," pp. 111-118 In N. R. Krieg and J. G. Holt (ed., Bergey's Manual of Systematic Bacteriology, Vol. I, Williams and Wilkins, Baltimore]. C. fetus shares a 16-30% DNA homology with C. hyointestinalis with which it is most closely related. C. hyointestinalis produces H.sub.2 S in triple sugar iron in contrast to C. fetus. The two species may also be distinguished via fatty acid profiles. C. hyointestinalis was first described in association with swine proliferative ileitis and has also been reported in health cattle and as an enteric pathogen of humans.
2. Description of the Prior Art
Nucleic acid hybridization using total genomic DNA has been used in the taxonomy [Fennel et al., J. Clin Microbiol 24:146-148 (1986); Steele et al., J. Clin Microbiol 22:71-74 (1985); Totten et al., J. Infect. Dis. 151:131-139 (1985); Von Sulffen, FEMS Microbiol Lett. 42:129-133 (1987); Chevrier et al., J. Clin. Microbiol. 27:321-326 (1989)] and in the diagnosis [Tomkins et al., Diagn. Microbiol. Infect. Dis. 4:71S-78S (1988) of Campylobacter. Nucleic acid probes have been developed for the genus Campylobacter [Freier et al., Clin. Chem. 34:1176 (1988)]; C. jejuni [Picken et al., Mol. Cel Probes 1:245-259 (1987); Korolik et al., J. Gen Microbiol. 134:521-529 (1988)]; C. hyointestinalis [Gebhart, J. Clin. Microbiol. 27: 2717-2723 (1989)]; and the C. coli--C. jejuni--C. laridis complex [Shrawder et al., Clin. Chem. 34:1176 (1988)].
Although the nucleotide sequences of ribosomal RNAs (rRNA) have been conserved through evolution, mutations have occurred as species diverge [Gray et al., Nucl. Acids Res. 12:5837-5852 (1984); Lane et al., Proc. Natl. Acad. Sci. U.S.A. 82:6955-6959 (1985); Woese et al., Microbiol. Rev. 47:621-629 (1983)]. Many of these changes exist in hypervariable regions. Oligonucleotides complementary to these regions have been synthesized which disciminate very closely related species. The 5S and 16S rRNA sequences of Campylobacter species have been examined for the purpose of studying the phylogeny and diversity of the genus, and partial sequences have been reported [Lau et al., Syst. Appl. Microbiol. 9:231-238 (1987); Romaniuk et al., FEMS Microbiol. Lett. 43:331-335 (1987); Paster et al., Intl. J. Syst. Bacteriol. 38:56-62 (1988); Tompson et al., Intl. J. Syst. Bacteriol. 38:190-200 (1988)]. Though deoxyligonucleotide probes specific for 16S rRNA have been reported for the genus Campylobacter [Moureau et al., J. Clin. Microbiol. 27:1514-1517 (1989); Rashtchian et al., Current Microbiol. 14:311-317 (1987); Romaniuk et al., 1987, supra; Wesley, J. Cell. Biochem. Suppl. 14C:182 (1990)] hypervariable regions between C. fetus and C. hyointestinalis have not been previously identified.